Purification and characterization of an alpha-mannosidase from the tropical fruit babaco ( Vasconcellea x heilbornii Cv. babaco).
An alpha-mannosidase (EC 3.2.1.24) present in the lyophilized latex of babaco ( Vasconcellea heilbornii ) has been purified to apparent homogeneity by native PAGE. The purification involves a three-step procedure with successive anion exchange with Q Sepharose HP, lectin affinity chromatography using ConA Sepharose 4B, and gel filtration using Superdex 200 prep grade. The molecular mass was determined to be in the range of 260-280 kDa by Superdex 200 prep grade gel filtration, and isoelectric focusing showed a pI range between 5.85 and 6.55, suggesting different glycosylated isoforms. The optimal temperature for the alpha-mannosidase was determined to lie between 50 and 60 degrees C, and the optimal pH was 4.5 at 50 degrees C. The K(m) value for p-nitrophenyl alpha-mannopyranoside (pNPM) was found to be 1.25 mM and the V(max), 2.4 microkat mg(-1) at 50 degrees C and 1.94 microkat mg(-1) at 40 degrees C. The pure alpha-mannosidase was specific for mannose and did not display activity for any other tested synthetic substrates.
Blom
Hans
H
Department of Physical and Analytical Chemistry, Uppsala University, Uppsala, Sweden. hans.blom@ytbioteknik.uu.se
Reyes
Fernanda
F
Carlsson
Jan
J
D000596:Amino Acids / Q000032:analysis; D031241:Caricaceae / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D004795:Enzyme Stability; D005638:Fruit / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008358:Mannose / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D043323:alpha-Mannosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D000596:Amino Acids; D002413:Cations, Divalent; D043323:alpha-Mannosidase; D008358:Mannose